Technologies for marking genes by the random integration of DNA sequences containing a detectable reporter (marker) are known and referred to as gene-trapping (Skarnes et al., 1992, Genes Dev., 6:903-18; Durick et al., 1999, Genome Res., 9:1019-1025; Pruitt et al., Development, 1997, 124:617-626). Gene-trap technologies provide vectors that typically do not contain a promoter. Instead, gene trap vectors provide specific sequences that generate fusion RNA transcripts when inserted into a gene. This makes gene trapping especially attractive for mammalian cells since these cells have complex genomic organization including large introns and small exons. The trapped gene can then be identified by mRNA sequence. Using these technologies it is possible to create libraries of cells in which the expression of the reporter gene provides measures of the activities of the genes (trapped genes) into which it is integrated Further, the genes that can be trapped represent the majority of genes within the cell.
Technologies are available for identification of trapped genes. In the gene-trap methods, identification of the trapped genes is based on the generation of fusion mRNA that contains the mRNA for the reporter protein. Subsequently, the gene in which the reporter gene has been inserted (the trapped gene) can be cloned by using standard techniques such as rapid amplification of cDNA (RACE).
For measurement of expression of the trapped genes, standard detecting techniques for the reporter gene have been used. For fluorescent reporters, such techniques include fluorescence activated cell sorting (FACS).
Libraries of trapped genes have been used to identify genes that respond to specific chemical compounds (Pruitt, 1992; Whitney, 1999, U.S. Pat. No. 5,928,888). Pruitt teaches inserting a β-galactosidase expression construct into a eukaryotic genome in a cell and contacting the cell with a chemical to detect changes in β-galactosidase activity. Whitney discloses inserting a beta lactamase expression construct into an eukaryotic genome in a cell and contacting the cell with a chemical to detect changes in the activity of beta lactamase. The prior art also provides for screening of a chemical compound for its ability to modulate gene expression, whereas the method of Whitney entails the use of FACS. However, there are limitations inherent to the prior art methods. First, although an individual chemical compound can be screened for an effect on multiple genes simultaneously, the method entails the preparative scale use of FACS to enrich for cells that respond to a given modulator over multiple rounds of enrichment. As a consequence, the method is prohibitively costly and time consuming to contemplate its use for high-throughput compound screens. Second, according to the method of Whitney, a library of cells containing trapped genes is divided into those that exhibit high levels of fluorescence and those that exhibit low levels of fluorescence, i.e. essentially on or off. FACS is used to enrich for cells that respond to a chemical compound by going from off to on, or on to off. Following the repeated rounds of enrichment discussed above, cells are recovered and the trapped genes are determined. Due to the inability of Whitney's method to resolve changes in gene expression less than that required to cause a cell to change from on to off, or off to on, qualitative changes in gene expression are not identified.
Accordingly, there is a need in the field of drug screening to develop methodologies whereby large number of compounds can be screened for their effects on the expression of genes and to develop methodologies whereby such effects can be determined by a quantitative measure.